Epigenetic changes caused by DNA methylation, histone modifications and histone variants play crucial roles in the regulation of chromatin structure and genome stability, developmental specific gene regulation, genomic imprinting and transcriptional silencing of transgenes and other foreign DNA. Cancers and many other diseases are associated with epigenetic alterations. The status of DNA methylation is determined by both methylation and demethylation reactions. DNA methylation by methyltransferase enzymes and the interplay between DNA methylation, small RNAs and histone modification patterns have been studied extensively. In contrast, the mechanism of active DNA demethylation and its relation with small RNAs and histone modifications are poorly understood. Our long-term goal is to understand the mechanism of active DNA demethylation and its role in epigenetic regulation. We have developed a unique system in the model organism Arabidopsis thaliana for dissecting DNA demethylation and other anti-silencing mechanisms. In this system, an active transgene and a homologous endogenous gene become silenced when cellular ROS (repressor of silencing) factors are mutated. ROS1 encodes a DNA glycosylase/lyase that prevents the hypermethylation of specific loci by active DNA demethylation through a base excision repair pathway. ROS2 encodes an adenosine phosphosulfate (APS) kinase, an enzyme involved in sulfur metabolism, which together with other data implicates sulfur modification of nucleic acids or proteins in preventing DNA hypermethylation. ROS3 encodes a 24-nt small RNA-binding protein that also functions in preventing DNA hypermethylation. We hypothesize that ROS1 functions in a DNA demethylase complex that includes ROS3 and other regulatory components, and small RNAs bound to ROS3 may guide the demethylase complex for locus-specific demethylation. To test this hypothesis, we propose the following studies: 1) Continue to characterize the biochemical properties of ROS1, investigate the physiological function of ROS1 and other related demethylases in environment-triggered DNA methylation changes, determine the mechanism of regulation of ROS1 transcription by genome methylation status, and test the function of ROS1-interacting proteins;2) Investigate the mechanism of ROS2 and ROS3 function in preventing DNA hypermethylation;and 3) Characterize the ros4 and ros5 mutants and isolate the ROS4 and ROS5 genes, which may encode additional components of the DNA demethylation pathway.